The long-term goal of this research is to gain a deeper understanding of the structure, catalytic mechanism and metabolic control of flavoproteins involved in fatty acid metabolism. Initially it is proposed to study mammalian acyl-CoA dehydrogenases, which participate in the first dehydrogenation step of Beta-oxidation. Despite their importance in cellular energy production, these flavoproteins have not been widely studied, largely because of insufficient amounts of the pure enzymes. A purification scheme using pig kidney has been developed which yields about l5-times more of an acyl-CoA dehydrogenase of general specificity per kg of tissue than previously obtained using pig liver. I wish to exploit this new method to further understanding of the acyl-CoA dehydrogenases. Purification of the physiological acceptor electron transferring falvoprotein from kidney will also be attempted to complement studies of the dehydrogenase. The dissociation constant and stoichiometry for binding various acyl-CoA derivatives to the dehydrogenase will be measured by spectrophotometric and gel filtration methods, and this may provide clues to the means by which the many intermediates of Beta-oxidation are efficiently handled by the enzymes of the sequence. Another aim is to identify catalytically essential residues via protein modification techniques, and to use suitable enzyme derivatives to probe the reaction mechanism. In particular, I hope to identify residues which activate the CoA-substrate prior to the flavin reduction step. Conventional reagents and CoA-thioesters bearing a reactive or latently reactive function in their acyl moieties will be tried. Once active site directed reagents have been incorporated into the dehydrogenase, it is hoped to develop methods for the exision of the CoASH moiety, using hydroxylamine under mild non-denaturing conditions, creating a new derivative which carries a smaller residual label. A procedure is also described for the rapid identification and purification of CoA-thioester labeled peptides via diagonal electrophoresis methods.